Eif4e gene mutations and potyvirus resistance

ABSTRACT

The invention concerns a method for obtaining potyvirus resistant plants exhibiting one or several mutations in a preserved region of the eIF4E translation factor, defined by the following general sequence (I): DX1X2X3X4KSX5QX6AWGSSX7RX8X9YTFSX10VEX11FWX12X13YNNIHX14P SKLX15X16GAD wherein:—X1, X2, X3, X4, X6, X7, X8, X9, X10, X12, X13, X15, et X16 represent each a neutral amino acid; X5 and X14 represent a basic amino acid; X11 represents an acid amino acid; D, K, S, Q, A, W, G, R, Y, T, F, V, E, N, I, H, P, and L have their usual one-letter code meaning.

The present invention relates to a method for selecting or obtainingpotyvirus-resistant plants. The method is particularly applicable toplants of the family of Solanaceae, Cucurbitaceae, Cruciferae andCompositae. The invention also comprises the sequences for conferringpotyvirus resistance and/or for labeling the genes for resistance or forsensitivity to these potyviruses.

The potyvirus group, the standard member of which is PVY, for potatovirus Y, is the largest plant virus group. In fact, potyviruses arecapable of infecting more than 30 plant families currently registered.This group comprises at least 180 members, which corresponds to a thirdof the plant viruses currently known. Potyvirus transmission is effectedby aphids (for example Myzus persicae) via the nonpersistent mode. Thesymptoms caused by potyviruses are abnormalities in leaf coloration(mosaics, yellowing of veins), leaf deformities, vein necroses which canresult in necrosis of the entire plant, and considerable decreases insize of the sick plant, greatly influencing productivity.

The Solanaceae, Cucurbitaceae, Cruciferae and Compositae areparticularly sensitive to potyviruses. The Solanaceae, and moreparticularly the tomato and the capsicum (or pepper), are infected withat least seven different potyviruses throughout the world: potato virusY (PVY) is present on all the growing areas, whereas the others areconfined to a continent (tobacco etch virus, pepper mottle virus andPeru tomato virus on the American continent, pepper veinal mottle virusand potyvirus E in Africa, and chili veinal mottle virus in Asia). Thiscompartmentalization is, however, no longer absolute, severalpotyviruses having been identified outside their area of origin. InFrance, and more particularly in the Mediterranean basin, thepredominant potyvirus is PVY. Having appeared in the 1970s, PVYepidemics developed in crops in the open field and then in crops undercover, where, from 1982, new PVY isolates were demonstrated, whichcaused particularly serious symptoms of necrosis in the tomato(Gëbre-Selassie et al., 1987). For some of these potyviruses, it ispossible to classify the isolates according to their ability tocircumvent resistance alleles. This is the case of PVY with respect tothe pvr2 gene in the capsicum, the only resistance gene that has beenused for a long time by selectors, but that is circumvented in theMediterranean region and in tropical regions. Despite the predominanceof PVY in France, the internationalization of the seed market makes itnecessary for selectors who sell their seeds abroad to use genescontrolling resistance to these various potyviruses. More generally,considering the economical importance of potyvirus infections and thelack of direct means for combating this type of infection, the searchfor resistant plant varieties constitutes one of the main lines of plantimprovement.

Potyviruses have a nonenveloped filamentous structure (Langenberg andZhang, 1997), from 680 to 900 nm long and from 11 to 15 nm wide(Dougherty and Carrington, 1988; Riechmann et al., 1992). The viralgenome consists of a single-stranded sense RNA approximately 10 kb inlength. The single-stranded RNA has a poly A tail at its 3′ end andbinds, in the 5′ position to a viral protein called VPg (Murphy et al.,1990, Takahashi et al., 1997). The viral RNA encodes 10 proteinsinvolved in polyproteins cleavage, genome replication, cell-to-cellmovement and long-distance movement, transmission via aphids, etc. Viruscontrol can only be carried out indirectly. Specifically, it is onlypossible to eliminate the vector of the disease (the aphids in thiscase) or to grow varieties resistant to the viral infection and/or tothe vectors.

Faced with an attack from a pathogen (viruses, bacteria, fungi ornematodes), the plant has several strategies for defending itself orwithstanding infection. Among the defense strategies, the plant can setup:

-   -   systems of mechanical defense by developing and reinforcing        physical barriers consisting of a thick cuticle on the leaves        and/or a deposit of callose or of lignins on the cell walls.        Thus, the entry of pathogens into and their movement in the        plant are made more difficult;    -   systems of chemical or biochemical defense by synthesizing toxic        compounds such as, for example, tannins, phytoalexins and        various protein complexes.

Among the resistance strategies, non-host resistance (when all theentities of a species are resistant to a given pathogen) isdistinguished from host resistance (when at least one entity of thespecies is sensitive to a strain of the pathogenic agent). Hostresistance, the most well-known and the most well-characterized to date,is that which involves a major, dominant gene. When the major gene is inthe presence of a gene specific for avirulence of the pathogenic agent,the incompatibility between the plant and the pathogen is set up and theplant is resistant. This interaction, described by Flor (1955), is alsocalled “gene-to-gene” model and is very often associated with localizednecrosis of the plant tissue at the site of infection (hypersensitivityreaction). Although quite widespread, this “gene-to-gene” model is notuniversal because some systems of resistance described do not functionaccording to this model, the differences lying in particular in the modeof action of the resistance gene. Genes exist which are recessive, whichare superdominant or which exert incomplete dominance. Severalavirulence genes can interact with the same resistance gene. Manyresistances are also polygenic, several genes present in the plant arethen involved in the resistance, each of them having a partiallyprotective effect and being able to control different mechanisms.

To date, many dominant genes according to the “gene-to-gene” model havebeen cloned. They have related gene structures, although they actagainst varied pathogenic agents (viruses, fungi, bacteria, insects,nematodes). The presence of conserved domains has made it possible todefine 4 major classes (Hammond-Kosack and Jones, 1997) of dominantgenes.

Singularly, it is estimated that 40% of potyvirus resistances arerecessive, whereas, in the other viral groups, this proportion onlyreaches 20% on average. Fraser (1992) has put forward the hypothesisthat recessive resistances are different from dominant resistances of“gene-to-gene” type and are the result of a specific deficiency oralteration in the product of a host's gene, necessary for accomplishingthe viral cycle in the plant. The dominant alleles for sensitivity aretherefore thought to correspond to the availability of this productinvolved in the plant/pathogen interactions.

It has been shown that point mutations in the viral gene encoding theVPg protein are involved in the circumventing of potyvirus resistance inseveral host-pathogen couples. This has been shown in the TVMV/Nicotianatabacum (va gene), PVY/tomato (pot-1 gene), LMV/lettuce (mol gene) andPSbMV/pea (sbm1 gene) couples (Keller et al., 1998, Morel, 2001,Redondo, 2001 Nicholas et al., 1997). This does not exclude the factthat other viral genes may also be involved.

Moreover, Wittman et al. (1997) have shown that an isoform of eukaryotictranslation initiation factor eIF4E from Arabidopsis thaliana interactswith the viral protein VPg of the turnip mosaic virus (TuMV). This sameinteraction has been detected between TEV VPg and eIF4E from tobacco andfrom tomato (Schaad et al., 2000).

The eIF4E gene encodes a eukaryotic RNA translation initiation factor.eIF4E corresponds to one of the subunits of the translation factor eIF4F(in wheatgerm, it corresponds to the p26 subunit). The eIF4E translationfactor binds to the mRNA cap at the m⁷Gs. The structure of eIF4E ischaracterized by a region rich in tryptophan residues (10 in Arabidopsisthaliana, 11 in wheat and 12 in mammals). These tryptophan residues arethought to be involved in binding to the m⁷G functional group (Rudd, K.et al. 1998). The eIF4E translation factor is encoded by a multigenefamily. For example, in Arabidopsis thaliana, 4 copies of eIF4E havebeen identified (Rodriguez et al., 1998, Robaglia et al., personalcommunication). These copies exhibit, in pairs, between 44 and 82%identity.

All these studies make reference to the correlation between theeIF4E/VPg interaction and the sensitivity of the plant to potyviruses,but none emphasize, or even suggest, that this interaction could resultin resistance. On the contrary, it is even indicated in Schaad et al.,2000, that the VPg/eIF4E interaction does not play a role in resistancesince the genetic determinants of the VPa/eIF4E interaction aredifferent from those which allow potyviruses (via VPg) to circumvent theresistance.

It is therefore to the inventors' credit, in such a state of the art, tohave demonstrated eIF4E proteins, and also the corresponding genes,involved in plant resistance or sensitivity to potyviruses.

The inventors have in particular noted that various potyvirus-resistantplants have point mutations located in the same region of the eIF4Eprotein; this region, which is very conserved between the eIF4E proteinsderived from various plant species, in particular from Solanaceae, isdefined by the general sequence (I) below:

-   DX₁X₂X₃X₄KSX₅QX₆AWGSSX₇RX₈X₉YTFSX₁₀VEX₁₁FWX₁₂X₁₃YNNIHX₁₄    PSKLX₁₅X₁₆GAD    -   in which:    -   X₁, X₂, X₃, X₄, X₆, X₇, X₈, X₉, X₁₀, X₁₂, X₁₃, X₁₅ and X₁₆ each        represent a neutral amino acid;    -   X₅ and X₁₄ represent a basic amino acid;    -   X₁₁ represents an acidic amino acid;    -   D, K, S, Q, A, W, G, R, Y, T, F, V, E, N, I, H, P and L have        their usual 1-letter code meaning.

The term “neutral amino acid” is here defined as any amino acid chosenfrom the following: alanine, valine, leucine, isoleucine, proline,tryptophan, phenylalanine, methionine, glycine, serine, threonine,tyrosine, cysteine, glutamine, asparagine. The term “charged amino acid”is defined as any amino acid chosen from the following: histidine,lysine, arginine, glutamate and aspartate. Among these charged aminoacids, histidine, lysine and arginine are basic amino acids andglutamate and aspartate are acidic amino acids.

The sequence (I) is also represented in the sequence listing in theappendix under the number SEQ ID NO: 1.

The mutations demonstrated by the inventors in the pepper are asfollows:

-   -   substitution of the neutral amino acid X₃ of sequence (I) with a        basic amino acid;    -   substitution of the neutral amino acid X₇ of sequence (I) with a        basic amino acid;    -   substitution of the aspartate residue in the C-terminal position        of sequence (I) with a neutral amino acid.

The mutation of X₃ was observed in pepper lines exhibiting two differenttypes of potyvirus resistance (pvr2¹ and pvr2²); the peppers exhibitingthe pvr2¹ phenotype also having the mutation at position X₇, and thepeppers exhibiting the pvr2² phenotype also having the mutation in theC-terminal position.

In the tomato, the inventors have in particular observed the followingmutations:

-   -   substitution of the neutral amino acid X₁ of sequence (I) with a        basic amino acid;    -   substitution of the Ala residue of the AWGSS unit of        sequence (I) with an acidic amino acid.

Due to the very high degree of sequence conservation of the eIF4E genesin eukaryotes and to the availability of 3D structure for the mouse andyeast eIF4E proteins (Marcotrigiano et al., 1997, Cell 89 : 951-961;Matsuo et al., 1997, Nat. Struct. Biol. 4 : 717-724), the positions ofthe mutations with respect to the 3D structure of eIF4E in the capsicumand the tomato can be determined. All these mutations are physicallyclose and at the surface of the protein. Moreover, these mutations donot involve amino acids that are very conserved in eukaryotes, nor thoseinvolved in the essential functions of eIF4E, namely cap recognition orinteraction between eIF4E and eIF4G or the 4E-binding protein.

However, it is probable that these mutations exert an effect on theVPg/eIF4E interaction by modification of the structure of eIF4E in theregion(s) thereof involved in this interaction. This structuralmodification probably results from the substitution of amino acids withamino acids having a different charge (replacement of neutral aminoacids with charged amino acids, or, conversely, of charged amino acidswith neutral amino acids, or amino acids having an opposite charge),which constitutes the point common to all the mutations demonstrated bythe inventors. It can therefore reasonably be assumed that othermutations of the same type in a plant eIF4E protein, in the regiondefined by sequence (I), will lead to similar structural modifications,producing the same effect on the VPa/eIF4E interaction.

In particular, it appears that the substitution of at least one of theneutral amino acids, X₁, X₂, X₃ or X₄ with a charged amino acid, inparticular with a basic amino acid, plays an important role in potyvirusresistance.

These observations makes it possible to propose tools, in particulargenetic tools, for screening and/or for obtaining plants resistant orsensitive to potyviruses.

The present invention relates more particularly to a method forselecting potyvirus-resistant plants, characterized in that it comprisesthe detection, in the plants to be tested:

-   -   of the presence or of the absence of an eIF4E protein        (hereinafter called: “wild-type eIF4E protein”) comprising a        region defined by sequence (I) above, or of a sequence encoding        said protein;    -   of the presence or of the absence of a mutant eIF4E protein        comprising a region derived from that defined by sequence (I)        above, by substitution of at least one neutral amino acid of        said sequence (I) with a charged amino acid, preferably a basic        amino acid, and/or substitution of at least one charged amino        acid of said sequence (I) with a neutral amino acid or an amino        acid having an opposite charge, or of a sequence encoding said        protein;    -   and the selection of the plants in which a mutant eIF4E protein        or a sequence encoding said protein is detected, and in which no        wild-type eIF4E protein or sequence encoding said protein is        detected.

A subject of the present invention is also a method for selecting plantswhich can be used for obtaining potyvirus-resistant plants,characterized in that it comprises the detection, in the plants to betested, of the presence or of the absence of the mutant eIF4E protein asdefined above or of a sequence encoding said protein, and the selectionof the plants in which said mutant eIF4E protein or a sequence encodingsaid protein is detected.

According to a preferred embodiment of the invention, said mutant eIF4Eprotein comprises a region derived from that defined by sequence (I)above, by:

-   -   a) substitution of at least one of the amino acids X₁, X₂, X₃ or        X₄ of said sequence (I) with a charged amino acid, and    -   b) substitution of at least one of the other neutral amino acids        of said sequence (I) with a charged amino acid and/or        substitution of at least one charged amino acid of said        sequence (I) with a neutral amino acid or an amino acid having        an opposite charge.

The detection of the presence or of the absence of a wild-type or mutanteIF4E protein can be carried out in particular using antibodiesspecifically directed against the desired form of the eIF4E protein.They may in particular be antibodies directed either against thewild-type form or against the mutant form of the region of eIF4E definedby sequence (I).

For the detection of the presence or of the absence of a sequenceencoding a wild-type eIF4E protein or of a sequence encoding a mutanteIF4E protein, many tools are available; they may in particular bepolynucleotides derived from the sequence of the eIF4E gene, and inparticular polynucleotides capable of hybridizing selectively eitherwith a wild-type allele or with a mutant allele of eIF4E, as definedabove, or polynucleotides which make it possible to amplify the regionof eIF4E containing the desired mutation; they may also be restrictionenzymes which recognize a target sequence present in the wild-type formbut not in the mutated form (or vice versa).

A subject of the present invention is thus the use of a selection toolchosen from:

-   -   a) a polynucleotide encoding a wild-type or mutant eIF4E protein        as defined above;    -   b) a polynucleotide complementary to the polynucleotide a);    -   c) an fragment of at least 10 bp of a polynucleotide a) or b);    -   d) an antibody directed against a wild-type or mutant eIF4E        protein as defined above;        for selecting potyvirus-resistant plants.

In particular, the invention relates to a method for selectingpotyvirus-resistant plants, characterized in that it comprises the useof at least one means of selection chosen from the group of genetictools (or similar) comprising:

-   *A/ all or part of one at least of the sequences selected from the    subgroup comprising:    -   SEQ ID NO: 2    -   SEQ ID NO: 4    -   SEQ ID NOs: 6 and 8,    -   any analog of these sequences resulting from the degeneracy of        the genetic code,    -   any cDNA sequence complementary to at least one of these        sequences and/or to at least one of their analogs;-   *B/ all or part of one at least of the products of transcription of    the sequences A;-   *C/ all or part of one at least of the products of translation of    the sequences A;-   *D/ all or part of at least one antibody specific for at least one    product of translation of C/;-   *E/ and any combination of tools A, B, C and D.

Preferably, the means of selection are selected from the tool subgroupsA/ and/or B/, and even more preferably from the tool subgroup A/.

By means of this simple, easy and reliable pinpointing of plantsresistant or sensitive to potyviruses, the inventors have thus developeda new method based on the use of sequences corresponding to the eIF4Egene.

The method which is the subject of the invention applies particularly tothe Solanaceae, Cucurbitaceae, Cruciferae and Compositae, and moreparticularly to plants of the genera Lycopersicon, Capsicum, Nicotiana,Solanum, Lactuca, Cucumis, Arabidopsis, etc.

The potyviruses concerned are, for example, potato virus Y (PVY),tobacco etch virus (TEV) and/or lettuce mosaic virus (LMV) and/orzucchini yellow mosaic virus (ZYMV) and/or turnip mosaic virus (TuMV).

To implement the method according to the invention, use is made ofnucleotide sequences and/or peptide sequences or restriction enzymes asmeans of detection, probes or primers, for selecting plants resistant orsensitive to potyviruses.

These means of detection comprise in particular nucleotide probes orprimers.

For the purpose of the present invention, the term “primer” is intendedto mean any polynucleotide sequence which can be used for amplifying asequence of an eIF4E gene liable to comprise a mutation associated withpotyvirus resistance. They are in particular polynucleotides which canbe used for amplifying all or part of the sequence of eIF4E encoding theregion of eIF4E defined by sequence (I), or of the mutant sequence whichis derived therefrom.

For the purpose of the present invention, the term “probe” is intendedto mean any polynucleotide sequence which hybridizes with a wild-typeeIF4E gene or with a mutant eIF4E gene as defined above. This includesin particular the nucleotide sequences capable of hybridizingselectively either with an allele of the eIF4E gene associated withpotyvirus resistance, or with an allele of the eIF4E gene associatedwith potyvirus sensitivity.

These probes and these primers can be used as markers specific for theplants resistant or sensitive to potyviruses.

In accordance with the invention, it is possible to sort thepotyvirus-sensitive plants from the potyvirus-resistant plants by meansof the genetic tools (or similar) (A) to (E), or even specificrestriction enzymes. The latter will be described below.

The nucleotide sequences (A) SEQ ID NOs: 2, 4, 6 and 8 correspond todifferent Solanaceae eIF4E genes involved in potyvirus resistanceencoding a eukaryotic RNA translation initiation factor. SEQ ID NO: 8corresponds to a recessive eIF4E allele for resistance to a potyvirus,while SEQ ID NOs: 2, 4, and 6 represent dominant eIF4E alleles forsensitivity to a potyvirus.

Means of selection or genetic markers for resistance or for sensitivityto potyviruses have therefore been discovered in accordance with theinvention. The method of selection according to the invention caninvolve, separately or together, the two types of means of selection ormarkers.

Naturally, the invention also encompasses all the equivalents to thesenucleotide sequences (A) SEQ ID NOs: 2, 4, 6 and 8, which conserve thefunction of eIF4E genetic marker for sensitivity/resistance topotyviruses specific to the reference sequences. As regards the DNAs,they are in particular the genetic degeneracy analogs and the cDNAsequences complementary to the reference sequences. The polynucleotideequivalents of the reference sequences (A) are also found among theirtranscription products (RNA) (B). The proteins (C) derived from (A) andfrom (B) constitute other intracellular markers for selecting plantsresistant or sensitive to potyviruses. Besides the targets (A), (B) and(C), the means of selection of the invention can also be nucleotideprobes capable of hybridizing with complementary nucleotide targets (A)and (B), or else protein-based means of detection (antibodies D) capableof pairing with specific antigenic targets (C). It is possible toenvision combining all these equivalent means (A), (B), (C) & (D) so asto form a selection tool (E).

The means according to the invention also cover any fragment of thesesequences (A), (B), (C) & (D). According to the invention, the term“fragment” is intended to mean:

-   -   either a polynucleotide of at least 10, 20, 30, 50, 100, 200,        300, 400, 500 contiguous nucleotides of the reference sequence;        preferred fragments are those which are capable of hybridizing        selectively, under stringent conditions, with said reference        sequence;    -   or a polyamino acid of at least 3, 6, 10, 15, 30, 60, 100, 150,        200 contiguous amino acids of the reference sequence; preferred        fragments are those which are capable of hybridizing        selectively, under stringent conditions, with said reference        sequence.

According to an advantageous embodiment of the invention, the method ischaracterized in that:

-   -   at least one means of detection comprising at least one of the        tools A, B, C, D, E as claimed in claim 1 and/or at least one        restriction enzyme is brought into contact with at least one        genomic and/or protein extract of a plant to be tested,    -   said genomic and/or protein extract, optionally paired and/or        hybridized and/or digested, is subjected to at least one        separation,    -   the possible pairings and/or hybridizations and/or digestions        liable to occur are revealed,    -   and the results are read so as to finally reach a conclusion as        to the presence or the absence of an allele for resistance        (pvr2¹) or of an allele for sensitivity (pvr⁺) to at least one        potyvirus.

This method falls within the framework of the methodologies known in thefield of the detection and recognition of plant genetic characteristics.

According to a first embodiment of the method, in which the principle ofselection is based on the use of one or more specific restrictionenzymes, the method can correspond to the following methodology:

-   -   the coding sequence of the eIF4E gene is amplified by PCR from        the DNA of the plant to be tested, for example using the primers        SEQ ID NOs: 18 and/or 19,    -   the amplification product is digested with a suitable        restriction enzyme,    -   the possible fragments obtained are separated,    -   and the resistant or sensitive plants are selected according to        the restriction profile for said amplification product.

For example, potyvirus-sensitive plants can be detected by means of arestriction profile which reveals the presence of a site for cleavage bythe TspRI enzyme or one of its isoschizomers, and potyvirus-resistantplants can be detected by means of a restriction profile which revealsthe absence of said site for cleavage with TspRI or one of itsisoschizomers and the presence of a site for cleavage with the MvnIenzyme or one of its isoschizomers.

According to a second embodiment of the method, corresponding to thecase where the mode of selection is the hybridization of complementarynucleotide sequences, the method preferably consists:

-   -   in extracting the DNA from plants,    -   in optionally subjecting this DNA to enzymatic digestion using        at least one restriction enzyme,    -   in denaturing the optionally digested DNA,    -   in bringing the DNA thus denatured into contact with a probe,        itself denatured beforehand and provided with at least one        label, so as to carry out the hybridization,    -   in eliminating the DNA and the nonhybridized probe,    -   in revealing the hybridization by means of the label,    -   and in selecting plants which have a hybridization profile        corresponding to cosegregation of the target hybridized with the        labeled probe and of the allele for resistance or for        sensitivity.

When the DNA has been digested with a restriction enzyme, thedistinction between the sensitive plants and the resistant plants can bemade by means of the difference in size of the hybridized fragments.

It can also be made using a probe capable of hybridizing selectivelywith the allele for resistance or the allele for sensitivity. Thehybridization of the single-stranded molecules of the probe and of thetarget is preferably carried out under stringent hybridizationconditions allowing selective hybridization, which can be determined ina manner known to those skilled in the art. In general, thehybridization and washing temperature is at least 5° C. below the Tm ofthe reference sequence at a given pH and for a given ionic strength.Typically, the hybridization temperature is at least 30° C. for apolynucleotide of 15 to 50 nucleotides and at least 60° C. for apolynucleotide of more than 50 nucleotides.

The level of signal generated by the interaction between the sequencecapable of hybridizing selectively and the reference sequences isgenerally 10 times, preferably 100 times, more intense than that of theinteraction of the other DNA sequences generating the background noise.

With a probe labeled, for example, with a radioactive element, such as³²P, or with a grafted enzyme, such as peroxidase, the hybridization isreadily revealed qualitatively and quantitatively.

The DNA used in the first or the second embodiment can be either totalDNA or cDNA.

According to a third embodiment (among others) of the method accordingto the invention, corresponding to the case where the mode of selectionis antibody/antigen pairing, the method preferably consists in detectingthe presence of a polypeptide partly consisting of all or part of one ofthe amino acid sequences described below and included in the invention.The method may consist in bringing the sample to be tested into contactwith an antibody as described above, and then in detecting theantigen/antibody complex formed.

Whatever the mode of selection, the method of selection according to theinvention is reliable and sensitive.

A subject of the present invention is also a polynucleotide encoding amutant eIF4E protein comprising a region derived from that defined bysequence (I) above, by substitution of at least one neutral amino acidof said sequence (I) with a charged amino acid, preferably a basic aminoacid, and/or substitution of at least one charged amino acid of saidsequence (I) with a neutral amino acid or an amino acid having anopposite charge.

According to a preferred embodiment, said polynucleotide encodes amutant eIF4E protein which comprises a region derived from that definedby sequence (I) above, by:

-   -   a) substitution of at least one of the amino acids X₁, X₂, X₃ or        X₄ of said sequence (I) with a charged amino acid, and    -   b) substitution of at least one of the other neutral amino acids        of said sequence (I) with a charged amino acid and/or        substitution of at least one charged amino acid of said        sequence (I) with a neutral amino acid or an amino acid having        an opposite charge.

Polynucleotides in accordance with the invention are, for example, thosewhich encode the variants of the sequences SEQ ID NO: 22 or 23associated with potyvirus resistance.

According to another of its aspects, the invention concerns a nucleotidesequence characterized in that it is described by a sequence chosen fromthe group comprising all or some of the following sequences:

-   -   SEQ ID NO: 2    -   SEQ ID NO: 4    -   SEQ ID NO: 6    -   SEQ ID NO: 8.

The nucleotide sequence SEQ ID NO: 2 is a cDNA sequence obtained fromtobacco DNA and corresponding to the tobacco gene. The nucleotidesequence SEQ ID NO: 4 is a sequence encoding an eIF4E protein of apotyvirus-sensitive variety of Lycopersicon esculentum. The sequencesSEQ ID NOs: 6 and 8 are sequences encoding eIF4E proteins from capsicum(Capsicum annuum), varieties Yolo Wonder and Yolo Y, respectively.

A subject of the present invention is also primers for amplifying aneIF4E gene, or a portion thereof, liable to contain at least onemutation, as defined above, associated with potyvirus resistance; theyare in particular primers for amplifying the sequence of eIF4E encodingthe region of eIF4E defined by sequence (I), or a mutant sequence whichis derived therefrom.

Primers in accordance with the invention can be readily defined by thoseskilled in the art, from the nucleotide or peptide sequences describedin the present invention.

By way of nonlimiting examples, mention will be made of: theamplification primers consisting of the nucleotide primer sequences SEQID NOs: 18 & 19; the cloning primers SEQ ID NOs: 10 to 17; the primersfor screening a BAC library, consisting of the nucleotide sequences SEQID NOs: 20 & 21.

SEQ ID NOs: 18 & 19 are primers derived from the coding sequence ofcapsicum Yolo Wonder eIF4E, which make it possible, in particular by PCRamplification and then by enzyme digestion, to detect the nucleotidesequences carrying the alleles for resistance, pvr2, and forsensitivity, pvr⁺, to potyviruses. The degenerate cloning primers SEQ IDNOs: 10 & 11 and the nondegenerate cloning primers SEQ ID NOs: 12 to 17were defined on the basis of an alignment of the eIF4E sequences fromtobacco, tomato and Arabidopsis and used for the synthesis (RACE) ofcDNA probes for detecting eIF4E in the tomato and capsicum genome. Theprimers SEQ ID NOs: 20 & 21 for screening a BAC library arenondegenerate. These primers SEQ ID NOs: 10 to 17, 20 & 21 canoptionally be used directly or indirectly (construction of selectiontools) in the detection of potyvirus resistance or potyvirus sensitivitycharacteristics.

A subject of the present invention is also a mutant eIF4E proteincomprising a region derived from that defined by sequence (I) above, bysubstitution of at least one neutral amino acid of said sequence (I)with a charged amino acid, preferably a basic amino acid, and/orsubstitution of at least one charged amino acid of said sequence (I)with a neutral amino acid or an amino acid having an opposite charge.

According to a preferred embodiment, said mutant eIF4E protein comprisesa region derived from that defined by sequence (I) above, by:

-   -   a) substitution of at least one of the amino acids X₁, X₂, X₃ or        X₄ of said sequence (I) with a charged amino acid, and    -   b) substitution of at least one of the other neutral amino acids        of said sequence (I) with a charged amino acid and/or        substitution of at least one charged amino acid of said        sequence (I) with a neutral amino acid or an amino acid having        an opposite charge.

The present invention also covers the products of translation of thenucleotide sequences SEQ ID NOs: 2, 4, 6 and 8, namely the polypeptideschosen from the group comprising all or some of the following sequences:

-   -   SEQ ID NO: 3    -   SEQ ID NO: 5    -   SEQ ID NO: 7    -   SEQ ID NO: 9.

The means for selecting potyvirus-resistance/sensitivity of plants,consisting of amino acid sequences, are preferably used as locatingtargets. They are then indirect selection means which underlie the useof specific means for detecting these peptide targets.

These means of detection are advantageously antibodies which constituteanother subject of the present invention. Thus, said antibodies arecharacterized in that they are specifically directed against all or partof one at least of the translation products C, and more particularly ofthe amino acid sequences SEQ ID NOs: 3, 5, 7, 9, 22 or 23 or a fragmentof at least 6 amino acids thereof. These antibodies may be monoclonal orpolyclonal.

The antibodies against the polypeptides as defined above can be preparedaccording to conventional techniques well known to those skilled in theart (for example, Kohler and Milstein, 1975; Kozbor et al. 1983,Martineau et al., 1998). An antibody according to the invention maycomprise a detectable isotope or nonisotope, for example fluorescent,label or else may be coupled to a molecule such as biotin according totechniques well known to those skilled in the art.

Another section of the invention relates to means of selection made upof probes for detecting plants resistant to at least one potyvirus,these probes being taken in themselves. In this section, probes fordetecting plants resistant to at least one potyvirus are defined.

A first category of probes is characterized in that each probe comprisesat least one sequence corresponding to all or part of SEQ ID NOs: 2, 4,6 and 8. Within this first category, the probes comprising at least onesequence corresponding to all or part of SEQ ID NOs: 2, 4, 6 and 8, andin particular to all or part of the portion encoding the region of theeIF4E protein defined by general sequence (I), are most especiallypreferred.

SEQ ID NO: 6 is a sensitivity probe derived from the capsicum YoloWonder. It differs from SEQ ID NO: 8, which is a resistance probederived from the capsicum Yolo Y, by virtue of two nucleotide bases.These mutations shown on SEQ ID NOs: 6 & 8 correspond to the restrictionsites TspRI for SEQ ID NO: 6 and MnvI for SEQ ID NO: 8, marking,respectively, potyvirus sensitivity in Yolo Wonder and potyvirusresistance in Yolo Y.

These probes are used to distinguish resistant and sensitive plants,either by selective hybridization and detection of the presence or ofthe absence of a hybridization signal, or by digestion with anappropriate restriction enzyme capable of differentially cleaving theallele for sensitivity and the allele for resistance, for example EcoRI,TspRI or MnvI, followed by hybridization of the probe with therestriction product. In the latter case, the sensitive plants aredistinguished from the resistant plants by the difference in size of thehybridized fragments.

The present invention also provides tools for carrying out anothermethod of selection in accordance with the first embodiment of themethod according to the invention, as defined above. According to thisfirst embodiment, a PCR amplification of the eIF4E sequence is first ofall carried out. The amplification is followed by selective digestionwith a restriction enzyme. The tools involved are therefore of twotypes: restriction enzyme(s) and PCR primer(s) for amplifying the eIF4Esequence.

A subject of the present invention is in particular a kit for detectingan eIF4E allele associated with potyvirus resistance or with potyvirussensitivity, characterized in that it comprises:

-   -   at least one restriction enzyme chosen from:    -   a) an enzyme which recognizes a restriction site I present in at        least one eIF4E allele associated with potyvirus sensitivity,        and absent from the eIF4E alleles associated with potyvirus        resistance;    -   b) an enzyme which recognizes a restriction site II present in        at least one eIF4E allele associated with potyvirus resistance,        and absent from the eIF4E alleles associated with potyvirus        sensitivity; and    -   a pair of nucleotide primers for amplifying eIF4E or a portion        thereof comprising the restriction site I and/or the restriction        site II.

For example:

-   -   for detecting an eIF4E allele associated with potyvirus        sensitivity, such as that represented by the sequence SEQ ID NO:        6, the restriction enzyme is TspRI, or one of its isoschizomers,        which recognizes a restriction site defined by the sense        sequence: NNCASTGNN{circumflex over ( )} and the antisense        sequence {circumflex over ( )}NNGTSACNN. The nucleotide primers        are chosen so as to allow amplification of the entire sequence        of eIF4E or of at least one portion thereof comprising the TspRI        site;    -   for detecting an eIF4E allele associated with potyvirus        resistance, such as that represented by the sequence SEQ ID NO:        8, the restriction enzyme is MvnI, or one of its isoschizomers,        which recognizes a restriction site defined by the sense        sequence: CG{circumflex over ( )}CG and the antisense sequence:        GC{circumflex over ( )}GC. The nucleotide primers are chosen so        as to allow amplification of the entire sequence of eIF4E or of        at least one portion thereof comprising the MvnI site.

In the two cases, the primers SEQ ID NO: 18 and SEQ ID NO: 19 may, forexample, be used.

As indicated above, the detection of plants sensitive or resistant topotyviruses can also be carried out by detection of the presence or ofthe absence of the wild-type or mutant form of the eIF4E protein.

Thus, the present invention encompasses the use of a wild-type or mutanteIF4E protein, as defined above, or of an antibody specific for one ofsaid proteins, for selecting potyvirus-resistant plants.

Preferably, said eIF4E protein is chosen from:

-   -   the protein represented by the polypeptide sequence SEQ ID NO:        3;    -   the protein represented by the polypeptide sequence SEQ ID NO:        5;    -   the protein represented by the polypeptide sequence SEQ ID NO:        7;    -   the protein represented by the polypeptide sequence SEQ ID NO:        9;    -   the group of proteins represented by the polypeptide sequence        SEQ ID NO: 22;    -   the group of proteins represented by the polypeptide sequence        SEQ ID NO: 23.

Thus, a category of means for detecting potyvirus resistance inaccordance with the invention is characterized in that each of thesemeans comprises at least one antibody specific for all or part of amutant eIF4E protein in accordance with the invention, and in particularfor a fragment of at least 6 amino acids thereof carrying a mutationassociated with the resistance, as defined above.

For example, a means for detecting the resistance may consist of anantibody specific for all or part of a polypeptide sequence as definedabove, in particular for a fragment of at least 6 amino acids thereofcarrying a mutation associated with the resistance.

The invention also relates to means for detecting potyvirus sensitivityor potyvirus resistance, each consisting of at least one amino acidsequence chosen from the group comprising the following sequences:

-   -   SEQ ID NO: 3;    -   SEQ ID NO: 5;    -   SEQ ID NO: 7;    -   SEQ ID NO: 9;    -   SEQ ID NO: 22;    -   SEQ ID NO: 23.

The invention relates more particularly to means for detecting potyvirussensitivity, each consisting of at least one antibody specific for anamino acid sequence chosen from the group comprising the followingsequences:

-   -   SEQ ID NO: 3;    -   SEQ ID NO: 5;    -   SEQ ID NO: 7;    -   SEQ ID NO: 9;    -   SEQ ID NO: 22;    -   SEQ ID NO: 23;    -   or a fragment of at least 6 amino acids of one of said        sequences, in particular a fragment of the region thereof        defined by sequence (I).

Preferably, each nucleotide probe or any other means of detectionmentioned above has at least one label, that is useful as an indicatorof the nucleotide hybridization or the antigen/antibody pairing at theheart of the detection of the sensitive sequence. Advantageously, thislabel is detectable by spectroscopic, photochemical, biochemical,immunochemical or else chemical means. For example, such a label mayconsist of a radioactive isotope of ³²P or ³H, of a fluorescent molecule(5-bromodeoxyuridine, fluoresceine, acetylaminofluorene) or else of aligand such as biotin. As regards more especially the nucleotide probes,the labeling thereof is preferably carried out by incorporation oflabeled molecules into the polynucleotides by primer extension or elseby addition to the 3′ or 5′ ends.

Preferably, the sequences used for detecting the potyvirus-resistantplants are used as nucleotide probes or primers.

It goes without saying that not all the abovementioned means ofdetection are limited strictly to the denoted sequences, but encompassall the equivalents consisting in particular of the similar sequenceswhich conserve the function under consideration, and as defined above.

Those skilled in the art are fully aware of the various methods forpreparing probes and primers, including by cloning and by the action ofrestriction enzymes, or else by direct chemical synthesis according totechniques such as the phosphodiester method of Brown et al. (1979) orthe solid-support technique described in European patent No. EP 0707592.The nucleic acids can be labeled, if desired, by incorporating adetectable molecule or label as set out above. Examples ofnonradioactive labeling of nucleic acid fragments are described inparticular in French patent No. FR 78 10 975 or else in the articles byUrdéa et al., (1988) or Sanchez-Pescador et al. (1988).

According to another of its aspects, the present invention relates tothe use of the means of detection defined above, for detecting plantsresistant/sensitive to at least one potyvirus.

In accordance with the invention, the MvnI and/or TspRI restrictionsites, which are incidentally known, of the eIF4E sequences are used asoligonucleotide marker(s) for potyvirus resistance/sensitivity.

Preferably, the restriction sites used as oligonucleotide marker(s)correspond:

-   -   to the sense sequence: CG{circumflex over ( )}CG and to the        antisense sequence: GC{circumflex over ( )}GC    -   and/or to the sense sequence: NNCASTGNN{circumflex over ( )} and        to the antisense sequence: {circumflex over ( )}NNGTSACNN.

The use of these restriction sites as markers (or labels or tags) forpotyvirus resistance on expressed sequences ties up with the firstembodiment of the method of detection described above, in which use ismade of restriction enzymes (for example: MvnI and/or TspRI) and ofprimers for amplification of the eIF4E sequence, for example: SEQ ID NO:18 and/or 19.

Considering the specificity of the MvnI & TspRI restriction sites, thepresent invention also encompasses the use, as oligonucleotide marker(s)for potyvirus resistance/sensitivity, of the abovementioned MvnI & TspRIrestriction sites and, preferably, of the restriction site correspondingto the sense sequence: CG{circumflex over ( )}CG and to the antisensesequence: GC{circumflex over ( )}GC and/or of the restriction sitecorresponding to the sense sequence: NNCASTGNN{circumflex over ( )} andto the antisense sequence: {circumflex over ( )}NNGTSACNN.

According to another of its aspects, the present invention relates to akit for selecting potyvirus-resistant/sensitive plants, comprising atleast one means of detection of antibody or polynucleotide type asdefined above. The kit comprises, where appropriate, the reagentsrequired for carrying out a hybridization or amplification reaction.

A subject of the invention is also the plants derived from the methoddescribed above and/or from the implementation of the tools and/or ofthe use and/or of the selection kit defined above. Preferably, theseplants belong to the family of the Solanaceae, Cucurbitaceae, Cruciferaeand Compositae. Even more preferably, they are chosen from tomatoes,capsicums and/or lettuce.

By way of examples, the inventors carry out the method which is thesubject of the invention by following the protocol for RFLP analysis(restriction fragment length polymorphism). To do this, the inventorshave used a conventional RFLP protocol in which the probes which are thesubject of the invention are labeled with ³²P and in which the DNA fromthe capsicum plants to be analyzed is digested with the EcoRIrestriction enzyme. At the end of this method, the inventors obtainhybridization profiles which are different between thepotyvirus-resistant plants and the potyvirus-sensitive plants, thusmaking it possible to select the sensitive or resistant plants. Thelatter may then enter into a program of plant improvement by successivecrossing.

The invention does not concern only the selection of plants resistant orsensitive to potyviruses. In fact, insofar as the inventors have beenable to identify the eIF4E gene determining a recessive resistance topotyviruses, it is from now on possible to envision obtaining newvarieties of genetically modified plants that are resistant (orsensitive) to at least one potyvirus.

The invention therefore relates to a nonbiological method for obtainingnew varieties of genetically modified plants that are resistant (orsensitive) to at least one potyvirus, characterized in that it consistsessentially in seeing to it that an eIF4E allele associated withresistance (or with sensitivity) to said potyvirus appears in the genomeof these plants, and/or in introducing said allele into the genome ofthese plants.

According to an advantageous embodiment of this method, the appearanceof the resistance allele is brought about by implementing a methodselected from the group comprising:

-   -   mutagenesis, advantageously “tilling”,    -   homologous recombination,    -   overexpression,    -   insertion/deletion,    -   gene silencing/transgenesis,    -   and combinations thereof.

The tools which may be used in the abovementioned nonbiological methodof production also form an integral part of the present invention.

A subject of the present invention is thus any constructed genetic unitcomprising a polynucleotide in accordance with the invention encoding amutant eIF4E protein, placed under the control of suitable elements forcontrolling the transcription and, optionally, the translation.

Said mutant eIF4E protein can advantageously be chosen from the variantsof the sequences SEQ ID NO: 22 or 23 associated with potyvirusresistance.

A subject of the present invention is also any constructed genetic unit,characterized in that it comprises:

-   -   at least one genetic tool A/ and/or B/ as defined above,    -   and/or at least one nucleotide sequence chosen from the group        comprising all or part of the following sequences:        -   SEQ ID NO: 6        -   SEQ ID NO: 8,    -   and/or at least one nucleotide sequence encoding an eIF4E factor        and comprising at least one MvnI and/or TspRI restriction site,        and preferably at least one restriction site corresponding to        the sense sequence: CG{circumflex over ( )}CG and to the        antisense sequence: GC{circumflex over ( )}GC and/or a        restriction site corresponding to the sense sequence:        NNCASTGNN{circumflex over ( )} and to the antisense sequence:        {circumflex over ( )}NNGTSACNN.

Another tool for genetic transformation covered by the inventionconsists of any vector for transforming plant cells, comprising at leastone constructed genetic unit as mentioned above. It may be any known andappropriate cloning vector (phages, plasmids, cosmids, etc.).

The plant cells and the microorganisms transformed by means of at leastone vector or of at least one constructed genetic unit as defined aboveare also part of the invention.

At a higher level, the invention encompasses the plants transformed bymeans of at least one vector and/or of at least one constructed geneticunit and/or of transformed plant cells and/or of transformedmicroorganisms, as they have been described above.

Those skilled in the art are very aware of all the direct or indirecttechniques for genetic modification. Additional details are given in theexamples which follow.

DESCRIPTION OF THE FIGURES

FIG. 1 represents the gel derived from a Southern blotting analysis andshowing the differences in profiles of the eIF4E marker for potyvirusresistance, observed for various sensitive or resistant capsicums. Thecapsicum genomic DNA is digested with the EcoRI enzyme and hybridizedwith the tobacco eIF4E cDNA—SEQ ID NO: 2—(example 3).

FIG. 2 represents the gel showing the PCR amplifications of the eIF4Egene involved in potyvirus resistance in capsicum, and demonstrates anMvnI restriction site which is differential between sensitive andresistant (example 4).

FIG. 3 represents the alignment of the eIF4E protein sequence fromvarious capsicum varieties that are sensitive or resistant topotyviruses.

FIG. 4 represents the alignment of the eIF4E protein sequence fromvarious tomato varieties that are sensitive or resistant to potyviruses.

EXAMPLES Example 1 Obtaining Tomato and capsicum Probes

The tomato and capsicum cDNAs were obtained by the 3′ and 5′ RACEtechnique (system for Rapid Amplification of cDNA Ends sold by thecompany Invitrogen™), from tomato and capsicum total RNA extraction, andusing degenerate primers defined on the basis of an alignment of theeIF4E sequences from tobacco, tomato and Arabidopsis. The 3′ portion ofthe cDNA was cloned by 3′RACE. Defined primers between the TAG and thepolyA tail of the sequences obtained by 3′RACE were used to obtain thecomplete cDNAs by 5′RACE. Primers used for the two steps of the 3′ RACE:Step 1: TCTAGATACAAYAATATCCAYCACCCAAGCAA = SEQ ID NO:10 Step 2:TCTAGATGGGRGCAGACTTTCAYTGTTT. = SEQ ID NO:11

The primers used for the three steps of the 5′RACE are illustrated intable I below: TABLE I Capsicum Tomato Step1 GTA TGA GAA ACT AAA AAA TGAGAA ACT AAA CTA = SEQ ID NO:12 CTA = SEQ ID NO:15 Step2 CAA CTT TTC AGTACG CTT TCC AGT ACG AAT AAT TGT GTT T = TGT GTT TCT T = SEQ ID NO:13 SEQID NO:16 Step3 TCC GAC ATT GCA TCA CTG CAT CAA GAA CTA TAC AGA ATT ATA C= GGT GTA A = SEQ ID NO:14 SEQ ID NO:17

Example 2 Assay for Hybridization and Selection of thePotyvirus-Resistant Plants (Resistance Controlled by the pvr2/pvr1/pvr5Locus)

Extraction of the DNA from the Plants to be Analyzed

The extraction of the DNA from the plants (Solanaceae, Cucurbitaceae,Cruciferae and Compositae) follows the protocols for standard extractionbased on the DNA microextraction protocol of Fulton and Tanksley, 1995

DNA Digestion and Separation on Agarose Gel

The protocol followed uses 2.5 U of enzyme/μg of DNA. The enzyme volumemust be less than 10% of the reaction volume. The reaction volume iscalculated as a function of the size of the well: it depends on the typeof tank and of comb used and on the gel volume (300 ml in general). Thevolume of the enzyme-specific buffer and the spermidine volume shouldeach represent 10% of the reaction volume:

-   -   ×μl DNA    -   1× buffer    -   1× spermidine (4 mM)    -   2.5 U of enzyme/μg of DNA    -   qs for H₂O reaction volume.

The digestion is carried out at 37° C. overnight. In parallel, samplesof λ phages digested with Hind III are prepared: 0.5 μg/well. Afterdigestion, correct digestion of the DNAs is verified on 1% agarose gel,1×TAE, with 1 μl of digestion product. If the digestion is correct, theloading buffer is then added. The loading buffer should represent aminimum of 10% of the total volume (or 20%). The sample is thendeposited onto a 300 ml, 1% agarose, 1×NEB, gel containing 10 μl of ETB.The migration is carried out at 25 V for 24 h in 1×NEB buffer (themigration is stopped at 2 cm from the edge of the gel).

Transfer onto Nylon Membrane

A Hybond N+ membrane and 1 Whatman paper are cut to the size of the gel.The gel is soaked for 30 min in a flat tank containing 1 L 0.25N HCl,with agitation (the blue becomes yellow).

During this time, the blotter is prepared by:

-   -   wetting a sheet of Whatman paper into 2×SSC and placing it on        the porous plate of the blotter;    -   and then by wetting the membrane and placing it on the Whatman,        which will be covered with the plastic screen.

The gel is rinsed in a tank containing distilled H₂O, and then placed onthe screen of the blotter, avoiding bubbles, and checking that thesystem is leaktight. The blotter is switched on at a maximum of 50 mb.Some 0.4N probe is poured over the gel. Two sponges soaked in sodiumhydroxide are placed on the gel, which will be covered with sodiumhydroxide until saturation is obtained.

The transfer is carried out in 2 h to 3 h. The membranes are rinsed in abath of 2×SSC for 10 to 15 min and then dried in the open air and bakedfor 2 h at 80° C.

Probe Preparation

Preparation of the probes by PCR labeling with ³²P involves probes of nomore than 3 kb, amplified by PCR or directly on plasmids, making itpossible to reveal the major bands for a probe of concentration between1 and 5 ng/μl.

The reaction conditions are summarized in table 2 below. TABLE 2 Finalconcentration H₂O 25.6 μl Promega 10× buffer 4 μl 1× Promega MgCl₂ 2.4μl Mix (50 μM ATG + 5 μM dCTP) * 2 μl 2.5 μM ATG; 0.25 μM dCTP 2 U/μlTaq 1 μl Primer (5 pM) 1 μl α32P-dCTP (1000 Ci/mmol, 3 μl 10 μCi/μl) DNAprobe 1 μl Final reaction volume 40 μl*: Mix (50 μM ATG + 5 μM dCTP) for labeling the RFLP probes by PCR.

Dilution of dATP, dTTP, dGTP to 10 mM, from the 100 mM stock solutions:

-   -   5 μl dNTP at 100 mM    -   45 μl H₂O.

Dilution of dCTP to 1 mM, from the 100 mM stock solution:

-   -   0.5 μl dCTP at 100 mM    -   49.5 μl H₂O.

Mix ATG+dCTP:

-   -   2.5 μl dATP at 10 mM, final concentration: 50 μM    -   2.5 μl dTTP at 10 mM, final concentration: 50 μM    -   2.5 μl dGTP at 10 mM, final concentration: 50 μM    -   2.5 μl dCTP at 1 mM, final concentration: 50 μM    -   490 μl H₂O.

The probes are labeled over the course of 30 PCR cycles of:

-   -   30 s at 94° C.    -   45 s at 52° C.    -   1 min 30 s at 72° C.

Once labeled, the probes are then denatured according to the followingprotocol:

-   -   each probe is added to a tube containing 160 μl of 0.8N NaOH+2        to 5 μl of labeled (by random priming) lambda    -   incubation is carried out for 5 min    -   neutralization is carried out with 200 μl of 1M Tris HCl.        Hybridization        Protocol According to Church and Gilbert (1984)        a—Prehybridization at 65° C. Overnight

20 ml of hybridization buffer are used per tube for 2 to 6 half-blots.Beyond this, 25 ml of buffer are used, without exceeding 10 half-blotsper tube. The membranes are wetted in a dish containing hybridizationbuffer before being slightly drained and then rolled up (altogether) andplaced in the tube.

During prehybridization, it is verified that the tubes are leaktight andthat the membranes unroll correctly, otherwise their direction ischanged.

* Composition of the Prehybridization and Hybridization Buffer:

For 500 ml: 21.91 g NaCl; 18.38 g sodium citrate; 380 ml H₂O; 15 ml 20%SDS; 25 ml 1M NaPO₄, pH 7.5; 25 ml 100× Denhardt's; 5 ml 0.25M EDTA; 50ml 50% dextran sulfate.

b—Hybridization at 65° C. for at Least 16 Hours

The temperature of the tubes is allowed to decrease before opening themin order to avoid wetting the thread. The denatured probe (5 min in 0.8MNaOH and then denaturation is stopped with 1M Tris-HCl) is added. Underthese conditions, the hybridization can last 48 or 72 hours.

c—Washing

The dishes (or trays) are washed in a large excess of buffer (1% SDS(Serva)) containing 40 mM NaPi, preheated to 65° C. For approximately5-10 half-membranes:

-   -   1 wash for 20 min at 65° C. with agitation. To wash, the        membranes are transferred one by one into a new tray containing        the preheated buffer. The radioactive washing buffers (at least        the first 2) are poured into a flask provided for this purpose;    -   1 rinse for 2-3 min in new buffer heated to 65° C.        d—Exposure

The membranes are dried on a bed of absorbent paper consisting of anarea of blue paper covered with white paper such as a roll of Torkpaper; they must not dry out. They are then placed in small plastic bagsfor the exposure, placed in a cassette with 1 intensifying screen.

According to the signal measured with a Geiger counter, they are exposedat −80° C. for a period of overnight to a few days.

e—Dehybridization of the Membranes Before Rehybridization

The membranes are dehybridized in a 0.1% SDS, 1 mM EDTA solution heatedto 80° C. (1 liter for 40 half-membranes) for 20 min at ambienttemperature. The membranes are then rinsed for 10 min in a 2×SSCsolution. Finally, the membranes are partially dried and then storeddamp in small plastic bags at 4° C.

Example 3 Correlation Between Potyvirus Resistance and eIF4E

The viral material used in these infection assays are the PVY N-605isolates obtained from Solanum tuberosum (Jakab et al., 1997), orPVY-LYE84 or PVY-LYE240r for tomato (Legnani et al., 1995) and thePVY-To72 and PVY-Si15 isolates for capsicum (Dogimont et al., 1996) andalso the TEV CAA-10 isolate (Legnani et al., 1996). The same protocol isused for all the other PVY and TEV isolates which are controlled by thepvr2/pyr1/pvr5 and/or pot-1 loci. The isolates are maintained accordingto the Bos procedure (Bos, 1969) and multiplied on Nicotiana tabacum cv.Xanthii plants before inoculation of the tomato or capsicum plants atthe stage of cotyledons with two leaves with horizontal blades. Theviral inoculum is prepared as described in the articles by Legnani etal. (1995, 1996) and by Dogimont et al. (1996). The cotyledons and thefirst two leaves of the plants are inoculated mechanically. The linesare evaluated under controlled conditions in a growth chamber (14 hoursof day time, 18° C. night and 24° C. day) in order to monitor theirreaction after inoculation. 4 weeks after inoculation, all the plantsare evaluated individually for the presence or the absence of the PVY orTEV capsid antigen using an ELISA assay (enzyme linked immunosorbentassay) as described by Legnani et al., 1995, 1996) and Dogimont et al.(1996). Other protocols which are entirely known to those skilled in theart can also be used for the mechanical inoculation of plants withpotyviruses.

The gel presented in FIG. 1 in the appendix shows the difference inprofiles observed between the eIF4E marker and the resistance topotyviruses controlled by the pvr2 locus. This complete cosegregationbetween potyvirus resistance and a copy of the eIF4E gene was observedon a linage by segregation of more than 500 plants.

The capsicum genomic DNA is digested with the EcoRI enzyme andhybridized with the tobacco eIF4E cDNA—SEQ ID NO: 2—(the same RFLPprofiles are obtained by hybridization with the tomato cDNA or thecapsicum cDNA).

The sensitive (S) plants possess the “lower” 7 kb restriction fragmentwhereas the resistant (R) plants possess the “upper” 7 kb restrictionfragments. The heterozygotes (Ht) plants exhibit the two restrictionfragments and are sensitive (because of recessive gene).

Example 4 Demonstration of eIF4E Mutations Associated with PotyvirusResistance

1) Demonstration of Differential Restriction Sites Between the Copies ofa Potyvirus-Sensitive capsicum Genotype and of a Resistant Genotype

Point mutations between the eIF4E gene of the capsicum variety “YoloWonder” (sensitive to potyvirus and carrying the pvr2⁺ allele) and thatof the capsicum variety “Yolo Y” (potyvirus resistant and carrying thepvr2¹ allele) were demonstrated by conventional sequencing techniques.Thus, at position 200, the coding sequence SEQ ID NO: 6 of eIF4E in YoloWonder exhibits a T, whereas the coding sequence SEQ ID NO: 8 of eIF4Ein Yolo Y exhibits an A. Similarly, at position 236, the coding sequenceof Yolo Wonder exhibits a T, whereas the coding sequence of Yolo Yexhibits a G.

The first point mutation corresponds to a TspRI restriction site (or itsisoschizomers) which exist only in Yolo Wonder. This differentialrestriction site was validated by PCR on the Yolo Wonder and Yolo YcDNA: definition of primers in the 5′- and 3′-position of the cDNA anddigestion of the PCR-amplified material with the TspRI enzyme.

(Same protocol as above for the MvnI enzyme, except that the digestionis carried out at 70° C. for this enzyme).

The second point mutation corresponds to an MvnI restriction site (orits isoschizomers) which exists only in Yolo Y. This differentialrestriction site was validated by PCR on the Yolo Wonder and Yolo YcDNA: definition of primers in the 5′- and 3′-position of the cDNA anddigestion of the PCR-amplified material with the MvnI enzyme. PCRreaction on the cDNA: sense primer: AAA AGC ACA CAG CAC CAA CA = SEQ IDNO:18 antisense primer: TAT TCC GAG ATT GCA TCA AGA A = SEQ ID NO:19

The reaction conditions are as given in table 3 below. TABLE 3 Finalconcentration H₂O 13.05 μl Promega 10× buffer 2.5 μl 1× Promega MgCl₂2.0 μl dNTP (4 μM) 1.25 μl 2 U/μl Taq 1 μl Primer (10 pM) 1.5 μl of eachcDNA (10 ng/μl) 3 μl Final reaction volume 25 μlAmplification cycles: 93° C.-3 min/35× (93° C.-45 s/53° C.-1 min/72°C.-2 min/72° C.-10 min

Digestion with the MvnI enzyme: 8 μl of PCR product+2 U of enzyme+1.3 μlof enzyme buffer+13.5 μl H₂O 2 h at 37° C. Migration on a 1×TAE 1.2%agarose gel.

The gel presented in FIG. 2 in the appendix shows the PCR amplificationsof the eIF4E gene involved in potyvirus resistance in the capsicum anddemonstrates an MvnI restriction site that is differential betweensensitive and resistant.

Band 1:

-   -   PCR-amplified material of the eIF4E gene of the        potyvirus-sensitive (S) capsicum Yolo Wonder-pvr2+ allele,    -   and absence of enzymatic digestion with MvnI.        Band 2:    -   PCR amplified material of the eIF4E gene of the        potyvirus-resistant (R) capsicum Yolo Y-pvr2¹ allele,    -   and demonstration of the MvnI restriction site.        Band 3:    -   1 kb ladder size marker.        2) Demonstration of eIF4E Mutations Associated with Potyvirus        Resistance

Sequencing of the eIF4E gene of various potyvirus-sensitive orpotyvirus-resistant capsicum varieties revealed mutations, associatedwith potyvirus resistance, in the same region of eIF4E.

Alignment of the eIF4E protein sequence of the various varieties isrepresented in FIG. 3.

Legend of FIG. 3:

-   YW=Yolo Wonder S/pvr2⁺-   DDL=Doux Long des Landes S/pvr2⁺-   PM1008=resistant to PVY(0)-   YY=Yolo Y/pvr2¹-   Avelar=pvr2¹ allele-   Vania=pvr2¹ allele-   PM994=resistant to PVY(0)-   Florida VR2=Florida/pvr2²-   C69=HD line derived from the F1 hybrid between CM334 and Yolo    Wonder/pvr5-   CM334=Criollo de Morelos 334/pvr5-   PM1014=resistant to PVY(0)-   Per=perennial/partial resistance (QTL) at the pvr2 locus-   Bold underlined=mutation common to all the resistants except PM1008-   Highlighted in gray=mutation specific to the pvr2¹ allele-   Bold not underlined=mutation specific to the pvr2² allele-   Underlined not bold=mutation specific to the pvr5 allele-   Highlighted in black=mutation specific to the PM1008 genotype.

These various variants are also represented in the sequence SEQ ID NO:22

Example 5 Demonstration of the Synteny Between capsicum and Tomato forthe Recessive Genes for Potyvirus Resistance (pot-1 Gene in Tomato andpvr2 Locus in capsicum)

Five main genes and several QTLs involved in potyvirus resistance aremapped on the capsicum genome. By means of the use of common RFLP probesfor mapping the genome and due to the highly conserved nature of orderof the markers between the tomato genome and the capsicum genome, thepotyvirus-resistance factors for capsicum are placed on the tomato map.The location of the capsicum potyvirus-resistance loci on the tomatochromosomes, and also that of the associated RFLP markers, isrecapitulated in table 4 with the references of origin. With the aim ofprecisely establishing the correspondence between the genomic regions ofcapsicum and of tomato with the potyvirus-resistance genes, the TG135and Cab3 RFLP markers are added to the pre-existing genetic linkage mapfor capsicum (Lefebvre et al., submitted). TABLE 4 ChromosomalAssociated position in Gene Spectrum markers^(b) tomato Reference pvr1TEV, TG56, 3 Murphy et al. 1998 PepMoV^(a) TG135 pvr2 PVY, CT31, 3Caranta et al. 1997 TEV^(a) TG132 Caranta et al., unpublished pvr3PepMoV^(a) nd^(c) nd^(c) Murphy et al. 1998 pvr4 PVY, CD72, 10 Carantaet al. 1999 PepMoV CT124 Grube et al. 2000 pvr5 PVY^(a) CT31 3 Carantaet al., unpublished pvr6 PVMV TG57 9 Caranta et al. 1996 pvr7 PepMoV,CD72, 10 Grube et al. 2000 PVY^(a) CT124^(a)Only the general spectrum of resistance is indicated for each gene,some of these resistance genes can be circumvented by virulent strains.^(b)The RFLP markers are obtained using randomly tomato genomic (TG) DNAor tomato leaf epidermis cDNA probes (CD and CT).^(c)nd = not determineda—AFLP and RFLP Labeling of the pot-1 Gene

The total DNA is extracted from approximately 1 g of fresh leaves fromF2 plants (Caranta et al., 1997).

The DNA samples from 6 F2 plants (derived from self-fertilization of theF1 hybrid between Lycopersicon esculentum Mospomorist and L. hirsutumPI247087) (pot-1⁺/pot-1⁺) having generated F3 families completelysensitive to PVY strain N 605 and the DNA samples from 9 F2 plantshaving generated F3 families completely resistant to potyvirus aregrouped together for a bulked segregant analysis and for AFLP tagging ofpot-1.

The AFLP markers are generated according to the protocol by Vos et al.(1995) with the EcoRI, HindIII and MseI restriction enzymes. The firstamplification is carried out using a combination of primers with asingle selective nucleotide and a second combination with 3 selectivenucleotides.

The AFLP markers associated with pot-1 are mapped on the lines producedfrom introgression of L. hirsutum into L. esculentum (Montforte andTanksley, 2001) in order to assign pot-1 to a tomato chromosome.

This assignation is validated by the mapping of RFLP markers located onthe target chromosome. The RFLP procedure is described bySaliba-Colombani et al. (2000). The screening of the polymorphismbetween Lycopersicon esculentum Mospomorist (sensitive to potyvirus) andL. hirsutum PI247087 (resistant to potyvirus) is carried out with 3restriction enzymes (EcoRI, HindIII and XbaI) and RFLP markerspre-mapped in tomato (CT, tomato cDNA derived from tomato epidermaltissue mRNA; TG, tomato genomic DNA clones; the CAB3 probe encoding achlorophyll a/b binding polypeptide, Tanksley et al., 1992). Thescreening makes it possible to map additional markers on chromosome 3.

Segregation analysis for the molecular markers (AFLP, RFLP) and for theresistance data are carried out using the Mapmaker/Exp v. 3.0 softwarewith a minimum Lod of 4.0 and a maximum recombination percentage of 0.3.The recombination percentage is then converted into mapping distance incentiMorgans (cM) using the Kosambi mapping function (Kosambi, 1944).

These results made it possible to locate the pot-1 gene for PVYresistance in tomato on chromosome 3 and to show that this gene isbordered by the same RFLP markers as the pvr2 locus in capsicum.

b—Mapping of eIF4E in Tomato

In parallel, the tomato eIF4E cDNA was mapped by the RFLP methoddescribed above on the lines produced from introgression of L. pennelliiinto L. esculentum (Eshed and Zamir, 1995). This study made it possibleto locate 5 copies of the eIF4E gene in tomato. One of these copies waslocated on chromosome 3, in the same genomic region as the pot-1 gene,thus confirming the synteny between capsicum and tomato for potyvirusresistance and, consequently, the possibility of using eIF4E as markersand tools for selecting resistance.

This demonstration of synteny between capsicum and tomato for therecessive genes for potyvirus resistance makes it possible to say that,if eIF4E is the resistance gene in capsicum, then eIF4E is also theresistance gene in tomato.

c—Cloning of a Tomato eIF4E Gene Associated with Potyvirus Resistance

cDNA of a tomato eIF4E gene similar to that isolated in capsicum wasisolated and cloned in tomato, according to the 3′ and 5′ RACE PCRmethod described in example 1; this gene was called eIF4E-2.

The coding sequence of this gene (variety ‘Mospomorist’ of L.Esculentum, sensitive to PVY and to TEV) is represented in the sequencelisting under the number SEQ ID NO: 2.

Point mutations between the eIF4E-2 gene of the L. esculentum‘Mospomorist’ and L. hirsutum PI134417 tomato genotypes (sensitive toPVY and TEV) and that of the L. hirsutum PI247087 genotype (resistant toPVY and to TEV, resistance controlled by the pot-1 recessive gene) weredemonstrated using conventional sequencing techniques.

The sequence alignment in represented in FIG. 4.

Legend: Mospo=Mospomorist; PI13=PI134417; PI24=PI247087

Bold and underlined: mutation observed only in PI247087; in bold notunderlined: mutation inter-specific between L. esculentum and L.hirsutum.

The eIF4E protein of L. hirsutum PI134417 and that of L. hirsutumPI247087 are also represented by the sequence SEQ ID NO: 23.

Example 6 Screening of the capsicum Genome BAC Library with PrimersDefined on the eIF4E Coding Sequence of the Yolo Wonder Genotype,Demonstration of the Cosegregation with Resistance and Determination ofthe Genomic Structure of the eIF4E Gene which Cosegregates with pvr2

A capsicum BAC library was constructed from a doubled haploid line HD208derived from the F1 hybrid of a cross between Capsicum annuum YoloWonder and C. annuum perennial. HD208 contains the pvr2+ dominant allelesensitivity.

The high molecular weight DNA was extracted according to the methoddescribed inhttp://www.ncgr.org/research/jag/papers00/paper300/indexpage300.html.The DNA was then partially and separately digested with threerestriction enzymes (EcoRI, BamHI and HindIII) in order to increase therepresentativeness of the entire genome. The digestive DNA was clonedinto the vector pCUGIBAC1.

The capsicum BAC library consists of 239 232 clones with an averageinsert size of 125 kb, which corresponds to a theoreticalrepresentativity of 10 genome equivalents (size of the capsicum genome3000 Mpb). This BAC library was organized into 623 DNA pools for thepurpose of screening by PCR (1 pool corresponds to a mixture of DNA from384 clones).

The following primers were defined on the coding sequence of eIF4E fromYolo Wonder: Pim1: 5′ AGA CTT TCA TTG TTT CAA GCA TAA 3′ = SEQ ID NO:20Pim4: 5′ GAT TAG AAA GTG CAA ACA CCA ATA C 3′. = SEQ ID NO:21

This pair of primers amplifies a 493 bp band on the cDNA and an 1800 bpband on the HD208 genomic DNA. This pair of primers was used to screenthe capsicum BAC library. Four BAC clones were identified carrying the1800 bp band (clones 27-BI, 5-2H, 111-4H and 184-4H).

These four BAC clones were digested with EcoRI and the restrictionprofiles show that they overlap and therefore clearly correspond to thesame locus. All the BAC clones reveal an EcoRI band of 7 kb, which wascloned into a vector pGEM3Zf. This 7 kb band, obtained by EcoRIdigestion, corresponds to that which co segregates with potyvirussensitivity (see example 3).

-   (1=clone 27-BI; 2=clone 5-2H; 3=clone 111-4H; 4=clone 184-4H)

The presence of the 1800 bp amplified material in the 7 kb fragmentconfirms that these four BAC clones carry the eIF4E gene correspondingto the cloned cDNA. Sequencing of the 7 kb insert made it possible todefine the size of the gene, which is 5500 bp, and to define theexon/intron structure: 5 exons and 4 introns.

Example 7 Experiment Comprising Transient Expression of the Yolo WondereIF4E cDNA in a Resistant capsicum Genotype (Carrying the pvr21 Allele)for Validation of the Role of eIF4E in Potyvirus Sensitivity

In order to validate the hypothesis that the pvr2+ sensitivity allelecorresponds to the Yolo Wonder eIF4E gene, experiments comprisingtransient expression of the Yolo Wonder eIF4E cDNA via a PVX (potatovirus X) viral vector (Chapman et al., 1992) are carried out on a Yolo Yresistant genotype, carrying the pvr2¹ allele.

The eIF4E cDNA derived from the Yolo Wonder sensitive genotype is clonedin an oriented manner into an expression vector PVX-CES-35S at the ClaIand SalI cloning site.

The Yolo Y resistant genotype (carrying the pvr2¹ allele) isco-inoculated with this recombinant plasmid and with the potato virus Y(PVY) pathotype 0. Transient expression of the eIF4E gene derived fromthe Yolo Wonder sensitive genotype via the recombinant PVX vector makesit possible for PVY to multiply in the resistant genotype. The PVY isdetected by the ELISA or RT-PCR method (Legnani et al., 1995, 1996,Dogimont et al., 1996).

The two genotypes of C. annuum Yolo Wonder and Yolo Y which havereceived the recombinant plasmid are sensitive to PVY: the virusesdetected by ELISA and RT-PCR on inoculated leaves and systemic leaves 10days after inoculation.

Similarly, eIF4E alleles derived from Yolo Wonder and Yolo Y, which areboth sensitive to TEV (tobacco etch virus), were expressed in aTEV-resistant capsicum genotype: Florida VR2. It is observed that thisexpression allows TEV to multiply (detected by ELISA and RT-PCR) in thisresistant genitor.

The tomato eIF4E-2 cDNA obtained from the variety Mospomorist (carryingthe sensitivity allele SEQ ID NO: 4) was also expressed according to thesame protocol in the Yolo Y resistant capsicum genotype.

Restoration of the sensitivity to PVY of the Yolo Y capsicum expressingthe tomato eIF4E-2 cDNA is also observed.

These results confirm the involvement of eIF4E in the sensitivity tovarious potyviruses, and also show that this system functions in aheterologous manner (tomato gene which functions in capsicum).

Example 8 Search for Mutants in the eIF4E Gene and in the Genes of theRNA Translation Initiation Complex for Creating Potyvirus-ResistantPlants

The members of the eIF4E multigene family belong to a complex of atleast 8 proteins which form the translation initiation complex ineukaryotic cells (Browning 1996).

The identification and the characterization of mutants in eIF4E and inthe other genes of the translation initiation complex, for creatingpotyvirus-resistant plants, take place in 4 steps and use a TILLINGsystem (Targeting Induced Local Lesions IN Genomes, McCallum et al.,2000):

-   -   (1) Generation of a collection of tomato mutants by chemical        mutagenesis. The genotype chosen is a microtomato, Lycopersicon        esculentum Microtom, which exhibits advantageous biological        characteristics (Meissner et al., 2000): sensitive to        potyviruses (PVY, TEV and PVMV), growth at high density (1000        plants/m²) and generation time of 3-4 months. The mutations are        obtained by chemical mutagenesis with ethylmethyl sulfonate        (EMS) (Koornneef et al., 1982): mutagenesis on 30 000 seeds,        sowing of the mutants and production of the M2 generation from        5000 M1 plants.    -   (2) Extraction of DNA from 20 plants per M2 family and formation        of DNA pools in 3 dimensions from a population of 100 000 M2        plants (5000 families).    -   (3) PCR amplification of the targeted genes and search for        mutations by denaturating HPLC. The sequencing of the genes        involved in the translation initiation complex are available on        the site http:/www.tigr.org/tdb/lgi. The PCR products are        subsequently denatured and then paired so as to allow the        formation of heteroduplexes. The mutations are then detected        either by denaturing HPLC (McCallum et al., 2000) or by means of        an enzyme which allows detection of “mismatches” in the        heteroduplex (CEL1 enzyme, Oleykowski et al., 1998).    -   (4) Characterization of the mutants in order to evaluate their        behaviour with respect to potyviruses: change from a sensitive        phenotype to a resistant phenotype. The procedure for        inoculation and detection of the potyviruses (potato virus Y,        tobacco etch virus, pepper veinal mottle virus) is identical to        that described in example 3.

Example 9 Creation of Potyvirus-Resistant Plants by Methods which MayInvolve Transgenesis

As an alternative to example 8, the resistance allele of the eIF4E gene(identified here in capsicum) or any other eIF4E allele which conferspotyvirus resistance (identified on the basis of examples 1 to 8) can betransferred, in planta, by means of methods such as site-directedmutagenesis (Hohn et al., 1999), or homologous recombination (Kempin etal., 1997), or by means of overexpression methods. In the overexpressionexperiments, the eIF4E allele which confers resistance is expressedunder the control of a strong promoter of the CaMV virus 35S type bytransgenesis in planta (Jones et al., 1992; Bevan 1984).

Resistant plants can also be created by knock-out of the endogenouseIF4E gene by means of gene silencing-type methods (post transcriptionalgene silencing) and the simultaneous expression, by transgenesis, of theeIF4E form which confers potyvirus resistance. A specific knock-out byPTGS can be carried out by directing it against the 5′ UTR of theendogenous eIF4E gene; the eIF4E form which confers resistance,expressed by transgenesis, will not carry the 5′ UTR sequence of theendogenous eIF4E. This specificity of the knock-out by PTGS against the5′ UTRs is based on the new data derived from the understanding of thePTGS mechanism (Nishikura 2001).

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1. A method for selecting potyvirus-resistant plants, comprising thedetection in the plants to be tested: of the presence or of the absenceof an eIF4E protein (hereinafter referred to as: “wild-type eIF4Eprotein”) comprising a region defined by SEQ ID NO: 1DX₁X₂X₃X₄KSX₅QX₆AWGSSX₇RX₈X₉YTFSX₁₀VEX₁₁FWX₁₂X₁₃YNNIHX₁₄PSKLX₁₅X₁₆GAD inwhich: X₁, X₂, X₃, X₄, X₆, X₇, X₈, X₉, X₁₀, X₁₂, X₁₃, X₁₅ and X₁₆ eachrepresent a neutral amino acid; X₅ and X₁₄ represent a basic amino acid;X₁₁ represents an acidic amino acid; D, K, S, Q, A, W, G, R, Y, T, F, V,E, N, I, H, P and L have their usual 1-letter code meaning, or of anucleotide sequence encoding said protein; of the presence or of theabsence of a mutant eIF4E protein comprising a region derived from thatdefined by sequence (I) by substitution of at least one neutral aminoacid of said sequence (I) with a charged amino acid, and/or substitutionof at least one charged amino acid of said sequence (I) with a neutralamino acid or an amino acid having an opposite charge, or of a sequenceencoding said protein; and the selection of the plants in which a mutanteIF4E protein or a nucleotide sequence encoding said protein isdetected, and in which no wild-type eIF4E protein or sequence encodingsaid protein is detected.
 2. A method for selecting plants which can beused for obtaining potyvirus-resistant plants, comprising the detection,in the plants to be tested, of the presence or of the absence of themutant eIF4E protein as defined in claim 1, or of a sequence encodingsaid protein, and the selection of the plants in which said mutant eIF4Eprotein or a sequence encoding said protein is detected.
 3. The methodas claimed in claim 1, wherein said mutant eIF4E protein comprises aregion derived from that defined by sequence (I), by: a) substitution ofat least one of the amino acids X₁, X₂, X₃ or X₄ of said sequence (I)with a charged amino acid, and b) substitution of at least one of theother neutral amino acids of said sequence (I) with a charged amino acidand/or substitution of at least one charged amino acid of said sequence(I) with a neutral amino acid or an amino acid having an oppositecharge.
 4. The method for selecting potyvirus-resistant plantscomprising contacting a candidate plant with a selection tool selectedfrom the group consisting of: a) a polynucleotide encoding a wild-typeor mutant eIF4E protein as defined in claim 1; b) a polynucleotidecomplementary to the polynucleotide a); and c) a fragment of at least 10bp of a polynucleotide a) or b); and determining the absence or presenceof an eIF4E protein.
 5. The method as claimed in claim 4, wherein saidplant is a member of a group selected from the group consisting ofSolanaceae, Cruciferae, Compositae, and Cucurbitaceae.
 6. The method asclaimed in claim 5, wherein said plant is a member of the groupSolanaceae.
 7. The method as claimed in claim 4, wherein eIF4E proteinis selected from the group consisting of: the protein represented by thepolypeptide sequence SEQ ID NO: 3; the protein represented by thepolypeptide sequence SEQ ID NO: 5; the protein represented by thepolypeptide sequence SEQ ID NO: 7; the protein represented by thepolypeptide sequence SEQ ID NO: 9; the group of proteins represented bythe polypeptide sequence SEQ ID NO: 22; and the group of proteinsrepresented by the polypeptide sequence SEQ ID NO:
 23. 8. The use asclaimed in claim 4, wherein said polynucleotide is selected from thegroup consisting of: the polynucleotide of sequence SEQ ID NO: 2; thepolynucleotide of sequence SEQ ID NO: 4; the polynucleotide of sequenceSEQ ID NO: 6; and the polynucleotide of sequence SEQ ID NO: 8; and alsothe polynucleotides complementary thereto, or the fragments of at least10 bp of said polynucleotides or complementary polynucleotides.
 9. Amethod for selecting potyvirus-resistant plants, comprising: digestingthe DNA extracted from a plant to be tested with a suitable restrictionenzyme; denaturing the digested DNA; contacting said denatured DNA witha probe consisting of a polynucleotide as defined in claim 4, providedbeforehand with at least one label, so as to carry out the hybridizationbetween said polynucleotide and said DNA; eliminating the DNA and theprobe which are not hybridized; detecting the hybridization by means ofthe label; selecting the plants which have a hybridization profilecorresponding to cosegregation of the target hybridized with the labeledprobe and of the allele for resistance or for sensitivity, the sensitiveplants being distinguished from the resistant plants by means of thedifference in size of the hybridized fragments.
 10. The method asclaimed in claim 9, wherein the restriction enzyme used is EcoRI.
 11. Amethod for selecting potyvirus-resistant plants, comprising: amplifyingthe coding sequence of the eIF4E gene by PCR from the DNA of the plantto be tested; digesting the amplification product with a suitablerestriction enzyme; separating the possible fragments obtained;selecting the resistant or sensitive plants according to the restrictionprofile for said amplification product.
 12. The method as claimed inclaim 11, wherein the potyvirus-sensitive plants are detected by meansof a restriction profile which reveals the presence of a site forcleavage by the TspRI enzyme or one of its isoschizomers, and thepotyvirus-resistant plants are detected by means of a restrictionprofile which reveals the absence of said site for cleavage by TspRI orone of its isoschizomers and the presence of a site for cleavage withthe MvnI enzyme or one of its isoschizomers.
 13. The method as claimedin claim 11, wherein the PCR amplification of the coding sequence of theeIF4E gene is carried out using, as primers, the oligonucleotides SEQ IDNO: 18 and SEQ ID NO:
 19. 14. A polynucleotide encoding a mutant eIF4Eprotein as defined in claim
 1. 15. The polynucleotide as claimed inclaim 14, wherein said polynucleotide is represented by the sequence SEQID NO:
 8. 16. A polynucleotide which can be used as a primer foramplifying by PCR a sequence encoding a plant eIF4E protein, whereinsaid polynucleotide is selected from the group consisting of: SEQ ID NO:18, and SEQ ID NO:
 19. 17. A kit for implementing a method as claimed inclaim 11, comprising: at least one restriction enzyme selected from thegroup consisting of: a) an enzyme which recognizes a restriction site Ipresent in at least one eIF4E allele associated with potyvirussensitivity, and absent from the eIF4E alleles associated with potyvirusresistance; and b) an enzyme which recognizes a restriction site IIpresent in at least one eIF4E allele associated with potyvirusresistance, and absent from the eIF4E alleles associated with potyvirussensitivity; and a pair of nucleotide primers for amplifying eIF4E or aportion thereof comprising the restriction site I and/or the restrictionsite II.
 18. The kit as claimed in claim 17, comprising: a pair ofprimers defined by the sequences SEQ ID NO: 18 and SEQ ID NO: 19; atleast one restriction enzyme chosen from TspRI or one of itsisoschizomers and MvnI or one of its isoschizomers.
 19. The method ofmarking an oligonucleotide sequence for potyvirus resistance orpotyvirus sensitivity comprising incorporating into said oligonucleotidesequence a MvnI restriction site preferably corresponding to the sensesequence: CG{circumflex over ( )}CG and to the antisense sequence:GC{circumflex over ( )}GC, and/or of a TspRI restriction site preferablycorresponding to the sense sequence: NNCASTGNN{circumflex over ( )} andto the antisense sequence: {circumflex over ( )}NNGTSACNN.
 20. Themethod for selecting potyvirus-resistant plants comprising contacting acandidate plant with a wild-type or mutant eIF4E protein, as defined inclaim 1, or of an antibody specific for one of said proteins.
 21. Themethod as claimed in claim 20, wherein said eIF4E protein is selectedfrom the group consisting of: the protein represented by the polypeptidesequence SEQ ID NO: 3; the protein represented by the polypeptidesequence SEQ ID NO: 5; the protein represented by the polypeptidesequence SEQ ID NO: 7; the protein represented by the polypeptidesequence SEQ ID NO: 9; the group of proteins represented by thepolypeptide sequence SEQ ID NO: 22; and the group of proteinsrepresented by the polypeptide sequence SEQ ID NO:
 23. 22. The mutanteIF4E protein as defined in claim
 1. 23. The mutant eIF4E protein asclaimed in claim 22, wherein said mutant eIF4E protein is selected fromthe group consisting of: the protein represented by the polypeptidesequence SEQ ID NO: 9; the variants of the protein represented by thepolypeptide sequence SEQ ID NO: 22; and the group of the proteinrepresented by the polypeptide sequence SEQ ID NO:
 23. 24. An antibodyspecifically directed against a mutant eIF4E protein as claimed in claim1, or a fragment of at least 6 amino acids of said polypeptide.
 25. Anonbiological method for obtaining plants resistant to at least onepotyvirus, comprising introducing an allele of the eIF4E gene associatedwith resistance to said potyvirus into the genome of said plants. 26.The method as claimed in claim 25, wherein the resistance allele isbrought about by implementing a method selected from the groupconsisting of: mutagenesis, advantageously “tilling”, homologousrecombination, overexpression, insertion/deletion, genesilencing/transgenesis, and combinations thereof.
 27. A constructedgenetic unit, comprising at least one polynucleotide as defined in claim4.
 28. A vector for transforming plant cells, comprising at least onegenetic unit as claimed in claim
 27. 29. A plant cell transformed withat least one vector as claimed in claim
 28. 30. A microorganismtransformed with at least one vector as claimed in claim
 28. 31. A planttransformed with at least one vector as claimed in claim
 28. 32. A plantcell transformed with at least one genetic unit as claimed in claim 27.33. A microorganism transformed with at least one genetic unit asclaimed in claim
 27. 34. A plant transformed with at least one geneticunit as claimed in claim
 27. 35. The method for selectingpotyvirus-resistant plants comprising contacting a candidate plant witha selection tool selected from the group consisting of: a) apolynucleotide encoding a wild-type or mutant eIF4E protein as definedin claim 3; b) a polynucleotide complementary to the polynucleotide a);and c) a fragment of at least 10 bp of a polynucleotide a) or b); anddetermining the absence or presence of an eIF4E protein.
 36. The methodas claimed in claim 35, wherein said plant is a member of a groupselected from the group consisting of Solanaceae, Cruciferae,Compositae, and Cucurbitaceae.
 37. The method as claimed in claim 36,wherein said plant is a member of the group Solanaceae.
 38. Apolynucleotide encoding a mutant eIF4E protein as defined in claim 3.39. The polynucleotide as claimed in claim 38, wherein saidpolynucleotide is represented by the sequence SEQ ID NO:
 8. 40. Themethod for selecting potyvirus-resistant plants comprising contacting acandidate plant with a wild-type or mutant eIF4E protein, as defined inclaim 3, or of an antibody specific for one of said proteins.
 41. Themethod as claimed in claim 40, wherein said eIF4E protein is selectedfrom the group consisting of: the protein represented by the polypeptidesequence SEQ ID NO: 3; the protein represented by the polypeptidesequence SEQ ID NO: 5; the protein represented by the polypeptidesequence SEQ ID NO: 7; the protein represented by the polypeptidesequence SEQ ID NO: 9; the group of proteins represented by thepolypeptide sequence SEQ ID NO: 22; and the group of proteinsrepresented by the polypeptide sequence SEQ ID NO:
 23. 42. The mutanteIF4E protein as defined in claim
 3. 43. The mutant eIF4E protein asclaimed in claim 42, wherein said mutant eIF4E protein is selected fromthe group consisting of: the protein represented by the polypeptidesequence SEQ ID NO: 9; the variants of the protein represented by thepolypeptide sequence SEQ ID NO: 22; and the group of the proteinrepresented by the polypeptide sequence SEQ ID NO:
 23. 44. An antibodyspecifically directed against a mutant eIF4E protein as claimed in claim3, or a fragment of at least 6 amino acids of said polypeptide.